The group of DNA-dependent nucleic acid polymerases is comprised of two subclasses of enzymes as follows; 1) the DNA-dependent DNA polymerases which are usually responsible for the replication of DNA genomes and 2) the DNA-dependent RNA polymerases. These polymerases play an essential role in the life cycle of all cells and many viruses. Agents that specifically inhibit the DNA and RNA polymerases of pathogenic organisms are potentially of therapeutic value in the treatment of disease. Inhibitors and activators of DNA-dependent RNA polymerases may also be useful as regulators of gene expression for purposes such as gene therapy. Current assays for inhibitors of DNA-dependent nucleic acid polymerases of the primer extension type work by measuring the effect of an agent on the amount of labeled nucleotide incorporated into a newly synthesized strand by extension of a primer on a DNA template. For a description of the primer extension method see Goff et al, Journal of Virology, 38, 239-248 (1981), Huang et al, The Journal of Biological Chemistry, 265, 11914-11918 (1990), and Orr et al, The Journal of Biological Chemistry, 267, 4177-4182 (1992). Assays for inhibitors and activators of DNA-dependent RNA polymerases may also be based on another method known in the field. Here, by placing under control of a relevant promoter, a gene coding for a protein which confers a discernible characteristic, candidate effectors of the polymerase are evaluated by their effect on the expression of this characteristic. The present invention also utilizes an operable linkage between the activity of a polymerase and the expression of a discernible characteristic, however, the mechanism employed by the present invention is unique and applicable to all classes of RNA and DNA-dependent nucleic acid polymerases.
The group of RNA-directed nucleic acid polymerases is comprised of two subclasses of enzymes as follows: 1) the RNA-dependent DNA polymerases (also referred to as the reverse transcriptases) and 2) the RNA-dependent RNA polymerases. These polymerases play an essential role in the life cycle of many vital pathogens. Two examples of human vital pathogens with an essential requirement for reverse transcriptase activity are Human Immunodeficiency Virus (HIV) and the Hepatitis B Viruses (HBV). Some examples of human viral pathogens with an essential requirement for RNA-dependent RNA polymerase activity are Polio myelitis virus, Human Reovirus, the Influenza viruses, and the Rhinoviruses. The reactions catalyzed by these polymerases are not known to occur in normal human cells and thus they are vital specific and considered good targets for therapeutic intervention by drug therapy. Screening assays which can rapidly and accurately detect the inhibitory activity of various agents toward specific RNA-dependent nucleic acid polymerases can therefore be very useful in the identification of new drug leads.
Much current attention is focused on the identification of inhibitors of the reverse transcriptase of HIV (HIV-RT). The target specific assay type in use is an in vitro primer extension assay in which the ability of an agent to inhibit reverse transcription is determined by comparing the amount of radiolabeled (or otherwise labeled) nucleotide incorporated into a reverse transcribed strand in the presence of the agent being tested versus that incorporated under the same conditions but in the absence of the agent. The RT used in the assay can be a purified product or lysates of HIV-infected cells may be used in which case the agent to be tested may be added to the cells before or after lysis. This assay can be automated for large scale screening but still contains multiple steps including the separation of the incorporated and unincorporated labeled nucleotides. Note that HIV-RT is also a DNA-dependent DNA polymerase.
RNA-template directed nucleic acid polymerases also play a vital role in viral pathogenesis in non-mammalian organisms including plants and in the normal biological function of various non-mammalian organisms including plants. Examples of the former case are various plant viruses with an essential requirement for RNA-dependent RNA polymerase function. An example of the latter case is the normal amplification of RNA by RNA-dependent RNA polymerases in plants. It is anticipated that inhibitors and activators of these polymerases will have uses in the fields of agriculture, biological control, gene control, and gene therapy.
The present invention provides a method for screening agents for their effect on the activity of specific RNA or DNA-dependent nucleic acid polymerases by operably linking the activity of such polymerases to novel template sequences which confer discernible characteristics. The present invention allows for rapid large scale screening of compounds by automation and both large and small scale screening by laboratories or individuals. The present invention also allows for the facile screening of agents and extracts in the non-laboratory field setting and is highly portable. The present invention drastically reduces the number of steps involved in the performance of an assay procedure compared to the primer extension assay method and can utilize a single assay reaction chamber throughout its implementation.
The present invention allows the direct colorimetric determination of the assay results. The assay method of the present invention can be wholly cell-based or comprise a simple in vitro assay system. The present invention makes no use of, nor does it generate, infectious viral particles or other pathogens.
The present invention also provides a novel method for controlling the expression of and, in general, the activity of a nucleic acid molecule. The ability to control the expression of exogenous genes is desirable for the application of gene therapy and for other purposes. One method, as described in Gossen and Bujard, Proc. Natl. Acad. Sci. USA, 89, 5547-5551 (1992), allows differential control of the expression of individual genes in mammalian cells in response to tetracycline by utilizing regulatory elements of the Tn10-specified tetracycline resistance operon of E. coli. Other methods described in the literature for eukaryotic gene control utilize various inducible promoters responsive to, for example, heavy metal ions, heat shock, and hormones. The present invention provides not only a method for controlling the expression of genes but also a method for controlling the activity of catalytic RNA and DNA molecules and other nucleic acid molecules.
The present invention utilizes the effects of antisense RNA and DNA on a complementary nucleic acid molecule. For a review on the subject, see Murray and Crocket, "Antisense Techniques: An Overview" in Antisense RNA and DNA, J. A. H. Murray, ed., Wiley-Liss Inc., 1-49 (1992). The present invention also utilizes the interaction between a polymerase and its respective polymerase binding site in a template molecule. The polymerase binding site and primer requirements for HIV-RT are disclosed in Weiss et al, Gene, 111, 183-197 (1992) and Kohlstaedt and Steitz, Proc. Natl. Acad. Sci. USA 89, 9652-9656 (1992). Those for HBV-RT are disclosed in Wang and Seeger, Cell, 71, 663-670 (1992).
Further objects and advantages of this invention will become apparent from a consideration of the drawings and ensuing description.